PROJECT SUMMARY/ABSTRACT Cellular diversity within a tumor is one of the main causes of cancer treatment failure. Presence of distinct subpopulations of cells with different sensitivity to any given therapy increases the chance of resistance and tumor recurrence. Therefore, understanding of mechanism governing the intratumor heterogeneity is vital for designing more effective regimens. Within the mentored phase of the proposal I am planning to investigate the influence of intratumor heterogeneity on HER2-targeted therapies in HER2+ breast cancer (Aim 1). In my preliminary study I used STAR-FISH, a novel method I developed allowing for simultaneous detection of point mutation and gene amplification at single cell level in intact tissue slides, to assess genetic heterogeneity and spatial distribution of subpopulations of cells in matched therapy nave and post- chemotherapy samples. We have found that changes in cellular diversity upon chemotherapy can predict patient outcome. Thus, in the current proposal I will test the hypothesis that emerged from those studies: intratumor heterogeneity could have a pronounced effect on targeted treatment. I will investigate this effect on samples of HER2+ breast cancers undergoing novel anti-HER2 regimen (Aim 1.A). I will also dissect the role of a point mutation in PIK3CA in epigenetic and phenotypic plasticity of HER2+ breast cancer cells (Aim 1.B). To uncover the mechanism that sustains intratumor heterogeneity in those tumors I will characterize the interactions between genetically distinct subpopulations (Aim 2). To accomplish the goals described in this proposal, I will use novel and unique techniques, validated in my previous studies. In the independent phase of the award I will concentrate my research efforts on highly aggressive brain tumors. These tumors display profound degree of intratumor heterogeneity and the current therapeutic interventions fail to significantly prolong lives of patients with this disease. Therefore, I am strongly motivated to study the genetic intratumor heterogeneity in gliomas (Aim 3). I will focus on the characterization of genetic diversity in the process of progression of low-grade glioma to glioblastoma and I will analyze targeted treatment-induced changes in intratumor heterogeneity and their influence on patient outcomes (Aim 3.A). I will also use a novel method to perform epigenetic profiling of genetically distinct subpopulations of glioblastoma cells to investigate the interplay between genetic and epigenetic heterogeneity (Aim 3.B). In summary, the proposed research will shed new light on therapeutic implications of intratumor heterogeneity and will help designing more effective cancer treatment. Throughout my scientific career I have demonstrated high productivity, devotion and strong technical skills, which are crucial for the successful execution of the proposed research. I have a long-term interest in the study of intratumor heterogeneity, as I have chosen the topic of cancer stem cells for my PhD thesis work and later joined the laboratory of Dr. Polyak at Dana-Farber Cancer Institute, specializing in breast cancer heterogeneity studies. As a postdoc, I have developed a new method allowing for assessment of genetic intratumor heterogeneity on single cell level in situ, as well as a new tool for automated image analysis. The application of this method on HER2+ breast cancer resulted in a manuscript entitled ?In situ single cell analysis identifies heterogeneity for PIK3CA mutation and HER2 amplification in HER2+ breast cancer?, which was recently published in Nature Genetics. During the mentored phase of the award I will expand those studies to investigate the mechanisms of clonal interactions and their changes upon novel targeted treatment. I will also acquire additional technical skills, including ChIP-seq and RNA-seq performance and computation, which are crucial to the experiments proposed in my independent research plan. Moreover, I will improve my management and leadership skills by participating in workshops within and outside DFCI. Dana-Farber Cancer Institute provides a nurturing environment for successful accomplishment of my scientific and career related goals. As one of the world's leading cancer research institutes and a part of the Harvard Cancer Center, DFCI facilitates collaborations between clinicians and scientists on local, national, and international level. Multiple seminars and workshops allow trainees to interact with experts to discuss research ideas and career development. The SPORE programs in breast and brain cancer will provide me with support of scientific leaders in research and treatment design. Excellent laboratory equipment and core facilities at DFCI will facilitate the completion of the proposed experiments. In addition to the institutional support, I will be provided with scientific and career development advice by the panel of mentors within Scientific and Mentoring Committee. The scientists supporting my efforts in this committee are all from distinct fields, yet they all share common interests and their diverse expertise will be invaluable to the advancement of the proposed project. My background, strong mentoring committee and excellent institutional environment of DFCI are key to the successful completion of the proposed aims. The results of the proposed study will broaden our understanding of the role of intratumor heterogeneity in therapy resistance, leading to improvements in new treatment design and better patient outcomes.